Organisms Diversity & Evolution (2018) 18:459–477 https://doi.org/10.1007/s13127-018-0376-4 ORIGINAL ARTICLE Anatomy of the Tantulocarida: first results obtained using TEM and CLSM. Part I: tantulus larva Alexandra S. Petrunina1 & Jens T. Høeg2 & Gregory A. Kolbasov3 Received: 21 December 2017 /Accepted: 16 August 2018 /Published online: 28 August 2018 # Gesellschaft für Biologische Systematik 2018 Abstract The morphology of Tantulocarida, a group of minutely sized ectoparasitic Crustacea, is described here using for the first time transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM). This enabled a detailed analysis of their internal anatomy to a level of detail not possible with previous light microscopic investigations. We studied the infective stage, the tantulus larva, attached on the crustacean host in two species, Arcticotantulus pertzovi and Microdajus tchesunovi, and put special emphasis on cuticular structures, muscles, adhesive glands used in attachment, sensory and nervous systems, and on the organs used in obtaining nutrients from the host. This allowed description of structures that have remained enigmatic or unknown until now. A doubly folded cuticular attachment disc is located at the anterior-ventral part of the cephalon and used for gluingthelarvatothehost surface with a cement substance released under the disc. Four cuticular canals run from a cement gland, located ventrally in the cephalon, and enter an unpaired, cuticular proboscis. The proboscis can be protruded outside through a separate opening above the mouth and is used for releasing cement. An unpaired and anteriorly completely solid cuticular stylet is located centrally in the cephalon and is used only for making a 1-μm-diameter hole in the host cuticle, through which passes a rootlet system used for obtaining nutrients. The rootlet system is a direct extension of the anterior gut of the tantulus, but inside the host, it consists of cuticle only. Several muscular systems seem to degenerate very quickly after the tantulus has settled on the host. The central nervous system is reduced to an absolute minimum in terms of both cell size and number. We discuss the morphology of the tantulus in light of the Tantulocarida being a sister group to the Thecostraca or even nested within that taxon. Keywords Tantulocarida . Thecostraca . Comparative anatomy . Phylogeny . TEM . CLSM Introduction mostly in the deep sea (Copepoda, Isopoda, Amphipoda, Ostracoda, Cumacea, Tanaidacea). Described as a class only The Tantulocarida is a group of minutely sized and ectopara- in 1983, tantulocarids have a truly enigmatic morphology and sitic Crustacea, infesting other meiobenthic crustaceans and remain one of the most poorly studied groups of arthropods. A tentative life cycle of the Tantulocarida has been reconstructed based on data gleaned from several different species (Huys * Alexandra S. Petrunina et al. 1993), but they have never been successfully reared in [email protected] the laboratory. The infective larval stage called the tantulus is one of the smallest multicellular organisms, but nevertheless it Jens T. Høeg [email protected] retains a general crustacean body plan (Olesen et al. 2014). This minute stage consists of a cephalon, six thoracic somites Gregory A. Kolbasov with paired natatory thoracopods, one limbless somite, and an [email protected] unsegmented abdomen (Figs. 1cand2). The cephalon lacks 1 Biological Faculty, Department of Invertebrate Zoology, Moscow any appendages, but has an oval-shaped, sucker-like attach- State University, Moscow, Russia ment organ located on its ventral side (Fig. 3) and also pos- 2 Department of Biology, Marine Biology Section, University of sesses a cuticular stylet for penetrating the cuticle of the host. Copenhagen, Copenhagen, Denmark In the currently accepted life cycle, the free-swimming 3 Biological Faculty, White Sea Biological Station, Moscow State tantulus eventually settles on the prospective host (Fig. 1), University, Moscow, Russia pierces the host cuticle, and starts feeding from it by means 460 Petrunina A.S. et al. Fig. 1 General morphology and rootlet system of the Tantulocarida (CLSM). Parthenogenetic female (a, b)and tantulus larva (c)of Arcticotantulus pertzovi on copepod host Bradya typica. a Parthenogenetic female attached to the host cuticle, general view, dorsally. b Cephalon and anterior part of the egg sac, a rootlet system originating from under the oral disc of the parasite. c Tantulus larva attached to the host cuticle with rootlet system at early stage of development. r.s., rootlet system; st., stylet; u.c., umbilical cord. Scale bars in micrometers of the rootlet system that enters through the hole (Fig. 1b, c). sexual stages. These form from the attached tantulus, but are The attached tantulus now passes through a complex meta- subsequently are released as free-living, non-feeding males morphosis leading to either a parthenogenetic female or to and females equipped with natatory thoracopods. After a pre- sexual male or female forms. The parthenogenetic female is sumably internal fertilization, the free female releases a new a sac-like stage that remains attached to the tantulus and thus generation of tantulus larvae. While already by itself complex, the host. It contains numerous eggs from which develop a new this hypothetical life cycle (Huys et al. 1993) is also chal- generation of infective tantulus larvae (Fig. 1a). The second lenged by the observation of a very aberrant nauplius stage type of metamorphosis results in a single male or female that does not fit into the currently accepted scheme (P. Fig. 2 General morphology of the tantulus larva. a Serratotantulus chetoprudae (SEM). b Arcticotantulus pertzovi (TEM, longitudinal section) Anatomy of the Tantulocarida: first results obtained using TEM and CLSM. Part I: tantulus larva 461 Fig. 3 Cephalon of the tantulus larva: general anatomy on longitudinal cross section. a Schematic drawing, nutrition holes in cuticle of rootlet system indicated by arrows. b TEM (Arcticotantulus pertzovi). a.d.pr., anterior disc of proboscis; br., brain; cem., cement; c.g., cement gland; c.g.c., cement gland cavity; c.g.d., cement gland duct; cut., cuticle; c.v., cuticular villi of the oral disc; ed., epidermis; g.c., gut cell; h.cut., host cuticle; np., neuropil; o.d., oral disc; ov., ovary/germinative cells; pr., proboscis; pr. c. op., proboscis cavity opening; r.s., rootlet system; st., stylet; st.r., stylet retractor. Scale bars in micrometers Martinez Arbizu, personal communication). It is therefore ap- two other groups of Thecostraca are also either obligatory parent that we still lack a satisfactory understanding of the life parasites (the Ascothoracida) or assumed to be so (the cycle for tantulocarids. Facetotecta), and the facetotectans even have a metamorpho- The tantulus fulfills the same role as the cypridoid larva sis, which rivals the rhizocephalans in complexity (Glenner in the hypothetically closely related Thecostraca by being et al. 2008). Obviously, therefore, it is of high interest to study responsible for attachment and metamorphosis into the en- the tantulus and compare it in detail with the infective stages suing stages. Furthermore, like the kentrogon stage in par- and general adaptation to parasitism in other Thecostraca. But asitic rhizocephalan cirripedes, the tantulus possesses a cu- unlike rhizocephalans, where the settlement and host infection ticular stylet, passes through a complex metamorphosis, has been studied in minute detail with transmission electron and the resulting parasite derives nutrients by means of a microscopy, the tantulus and all other stages of the system of rootlets inside the crustacean host. On the other tantulocarid life cycle has until now been examined by light hand, the rhizocephalan parasite is first injected as an en- microscopy only. These studies have revealed a lot of struc- tirely endoparasitic stage that only later emerges on the tural details, but they also raise many questions and are in surface of the host, while the tantulocarid parasite remains general very difficult to compare with TEM level information. external except for the rootlets. A very preliminary TEM study of the tantulus was given by The apparently close relationship between the Petrunina et al. (2014). The purpose of this paper is therefore Tantulocarida and the Thecostraca and the similarities be- to provide a comprehensive description of the anatomy of the tween the tantulus and the rhizocephalan kentrogon obviously tantulus larva using both TEM and CLSM techniques. Based raise the question whether the two groups could be closely on this, we will compare the results with previous light micro- related or whether tantulocarid parasitism evolved scopic accounts and thereby provide a platform for discussing convergently. It adds to the complexity of this problem that tantulocarid relationships and evolution. 462 Petrunina A.S. et al. Materials and methods dorsally in one specimen (Fig. 5e) and by position could possibly have belonged to a stylet retractor (Fig. 3a). The cuticle varies in Two species of the Tantulocarida Arcticotantulus pertzovi thickness from 0.20 to 0.70 μm and is clearly differentiated into a Kornev, Tchesunov, Rybnikov, 2004 and Microdajus thin electron-dense and 20-nm-thick epicuticle and homogenous tchesunovi Kolbasov et Savchenko, 2010 are known to para- procuticle (Figs. 5f, g, 3,and4). Procuticle can consist of up to sitize a harpacticoid Bradya typica Boeck, 1873 and a tanaid five irregular layers of different density (Fig. 5g). Typhlotanais sp. that are common meiobenthic crustaceans inhabiting silty sediments of the Kandalaksha Bay in the vi- Thoracic somites and muscular system cinity of the White Sea Biological Station (66°31′41″ N, 33°11′08″ E). Bottom mud was obtained from the depths of Most of the thorax and the thoracopods are filled with a pop- 20–40 m using light hyperbenthic dredge. A standard bub- ulation of small-sized cells with a high nucleus-to-cytoplasm bling or flotation method was applied to extract meiobenthic ratio. The nuclei contain highly condensed chromatin and crustaceans from the silt (Higgins 1964).